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. 2009 Sep 2;106(38):16174–16179. doi: 10.1073/pnas.0901455106

Fig. 3.

Fig. 3.

Role of ATP and the CBS domain of SpoIVFB in cleavage of Pro-σK. (A) The C-terminal 10 residues of SpoIVFB are important for cleavage of Pro-σK. E. coli cells bearing pZR12 to produce Pro-σK (1–126)-His6 and either pZR209 to produce wild-type (WT) TM-SpoIVFB (lane 1) or pZR244 to produce mutant TM-SpoIVFB lacking the C-terminal 10 residues (CΔ10) (lane 2), were collected 2 h after IPTG induction. Extracts from equivalent cell amounts were immunoblotted using antibodies against FLAG (top panel) or penta-His (bottom panel). (B) The CBS domain of SpoIVFB binds ATP. The Immobilon-P membrane was subjected to autoradiography to detect [α-32P]ATP bound to protein (lane 3), then stained with Coomassie blue to detect His10-CBS (lane 2) and marker proteins (lane 1). (C) ATP affects the interaction of Pro-σK with the CBS domain of SpoIVFB. Cobalt-affinity chromatography of His10-CBS and coexpressed Pro-σK (1–126)-FLAG2 in the absence (lane 1) or presence (lane 2) of ATP as indicated with the flow-through (unbound) and high imidazole eluate (bound) immunoblotted using antibodies against penta-His and FLAG to detect His10-CBS and Pro-σK (1–126)-FLAG2, respectively.